Exploring volatile general anesthetic binding to a closed membrane-bound bacterial voltage-gated sodium channel via computation

Abstract

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics "flooding" simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the "fenestrations" present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.

Figure 1. Simulation setup and isoflurane binding sites.

(A) Structural domains of NaChBac showing the VSD (purple), the S4–S5 linkers (red) and the pore domain (blue/green) with the S6 helices highlighted in green. (B) Isoflurane flooding simulation system initial setup showing the NaChBac protein (orange) in the POPC bilayer (white) with isoflurane molecules in the aqueous phase. (C) The three binding sites identified by clustering analysis: extracellular site (red), linker site (yellow) and cavity site (purple/green).

Figure 2. Isoflurane binding sites.

Extracellular (A and B) and linker site (C and D): residues interacting with isoflurane are highlighted in the insets B and D. For clarity, alternating subunits are shown in cyan/pink and labeled a through d. (E and F) Side and top views of isoflurane molecules occupying the cavity site and fenestrations.

Figure 3. Isoflurane displacement of cavity water molecules.

(A) Absolute number of water molecules (red) and isoflurane molecules (blue) within the cavity as a function of time. (B) Number of cavity waters as a function of the number of bound isoflurane molecules. Note that each isoflurane molecule displaces approximately 6 waters.

Figure 4. Fenestrations as the hydrophobic access pathway.

A and B show two views of an isoflurane molecule diffusing in (A) and out (B) of the central cavity through two adjacent fenestrations. Cavity and fenestrations are shown as grey surfaces.